INCREASED WHEAT (TRITICUM AESTIVUM L.) GRAIN HARDNESS …

INCREASED WHEAT (TRITICUM AESTIVUM L.) GRAIN HARDNESS

CONFERRED BY NOVEL PUROINDOLINE HAPLOTYPES AND ITS EFFECTS ON

END USE QUALITY

by

Nicholas Paul Reynolds

A thesis submitted in partial fulfillment

of the requirements for the degree

of

Master of Science

in

Plant Science

MONTANA STATE UNIVERSITY

Bozeman, Montana

November 2009

©COPYRIGHT

by


2009

All Rights Reserved

ii

APPROVAL

of a thesis submitted by


This thesis has been read by each member of the thesis committee and has been

found to be satisfactory regarding content, English usage, format, citation, bibliographic

style, and consistency and is ready for submission to the Division of Graduate Education.

Dr. Michael J. Giroux

Approved for the Department of Plant Science

Dr. John Sherwood

Approved for the Division of Graduate Education

Dr. Carl A. Fox

iii

STATEMENT OF PERMISSION TO USE

In presenting this thesis in partial fulfillment of the requirements for a

master‟s degree at Montana State University, I agree that the Library shall make it

available to borrowers under rules of the Library.

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only by the copyright holder.


November 2009

iv

ACKNOWLEDGMENTS

This research would not be possible without the guidance and immense amount of

patience shown to me by my advisors Dr. John M. Martin and Dr. Michael J. Giroux, as

well as my committee. Most importantly, I would like to give appreciation to those in my

life that have supported me most throughout the last two and a half years; Kacey,

Duncan, Juneau, and McKinley are the joys in my life and make me believe that I can do

anything. Without them, I may still have a couch without a giant hole in it or get to enjoy

a full night‟s sleep, but I wouldn‟t be nearly as complete. Thank you to everyone for your

ongoing support and love.

v

TABLE OF CONTENTS

1. INCREASED WHEAT (TRITICUM AESTIVUM L.) GRAIN HARDNESS

CONFERRED BY NOVEL PUROINDOLINE HAPLOTYPES FROM AEGILOPS

TAUSCHII ........................................................................................................................ 1

Introduction ..................................................................................................................... 1

Materials and Methods .................................................................................................... 4

Plant Material .............................................................................................................. 4

Selection of Segregating Lines .................................................................................... 8

Field Testing ................................................................................................................ 8

RNA expression and qRT-PCR ................................................................................... 9

Total TX-114 and Friabilin Levels ............................................................................ 10

Grain hardness and Seed Size Traits ......................................................................... 12

Data Analysis ............................................................................................................. 12

Results ........................................................................................................................... 13

Plant and Genetic material ......................................................................................... 13

Pina and Pinb Transcript Levels ............................................................................... 15

PINA and PINB Protein Expression Levels .............................................................. 18

Grain Texture ............................................................................................................. 23

Discussion ..................................................................................................................... 29

Conclusion ..................................................................................................................... 33

2. NOVEL HA LOCUS, PINA-D1C/PINB-D1H, IMPACTS SOFT WHITE SPRING

WHEAT MILLING AND BAKING CHARACTERISTICS........................................ 34

Introduction ................................................................................................................... 34

Materials and Methods .................................................................................................. 36

Plant Material ............................................................................................................ 36

Selection of Segregating Lines .................................................................................. 36

Field Testing .............................................................................................................. 37

End Quality Testing ................................................................................................... 37

Data Analysis ............................................................................................................. 39

Results ........................................................................................................................... 39

Discussion ..................................................................................................................... 49

Conclusion ..................................................................................................................... 51

LITERATURE CITED ..................................................................................................... 53

APPENDIX A: Backcross Population Agronomic Data ............................................... 59

vi

LIST OF TABLES

Table Page

1. Synthetic Hexaploid Genetic Backgrounds ....................................................................6

2. Pina, Pinb, and Actin Primers ........................................................................................7

3. Pin Transcript Levels ...................................................................................................17

4. PIN Protein Levels .......................................................................................................22

5. Single Kernel Characterization System (SKCS) and segregation data of

BC2F2 and BC3F2 derived populations from elite synthetic line

#14 cross with Recurrent Parents „Alpowa‟ and „Vanna‟ .............................................25

6. Single Kernel Characterization System (SKCS) and segregation data of

F2 populations ...............................................................................................................26

7. Hardness Differences Based on Haplotype ...................................................................28

8. Grain Hardness, Seed Weight, and Protein Content ....................................................41

9. Mean bran and flour yields after milling and flour ash content ...................................43

10. Flour Particle Size Distribution ...................................................................................44

11. Solvent Retention Capacities ........................................................................................45

12. Cookie Baking Quality .................................................................................................47

vii

LIST OF FIGURES

Figure Page

1. Deduced Amino Acid Sequences of Puroindoline-a and Puroindoline-b .................15

2. TX-114 Extracted Endosperm Puroindolines ............................................................20

3. Friabilin SDS-PAGE ..................................................................................................21

4. Cookie Top Grain Quality .........................................................................................48

..

viii

ABSTRACT

The Puroindoline genes (Pina and Pinb) together comprise the wheat (Triticum

aestivum L.) Hardness locus (Ha) located on chromosome 5D and control grain texture.

While hard wheats contain a mutation in either Pina or Pinb, there is no puroindoline

allelic diversity among soft hexaploid wheats as all tested soft wheats carry the Pina-

D1a/Pinb-D1a alleles. However, abundant Pina and Pinb allelic variation exists within

synthetic hexaploid wheats created using novel D genome donors. A previous study

indicated that the Pina-D1c/Pinb-D1h haplotype conferred a 10 unit decrease in hardness

compared to the Pina-D1a/Pinb-D1a Ha haplotype. Here we tested the effects of four

novel Ha locus haplotypes (Pina-D1c/Pinb-D1h, Pina-D1e/Pinb-D1i, Pina-D1a/Pinb-

D1i, and Pina-D1j/Pinb-D1i) from synthetic wheats by crossing Pina-D1c/Pinb-D1h into

the soft white spring wheat, „Alpowa‟ and all four haplotypes into the soft white spring

wheat, „Vanna‟. PINAa/PINBh, PINAc/PINBh, and PINAj/PINBh did not affect protein

levels and Pina-D1c/Pinb-D1h did not affect transcript levels found in the seed. Pina-

D1c/Pinb-D1h was found to increase grain hardness relative to the wild type Ha locus by

an average of 6.5 units for all populations, Pina-D1e/Pinb-D1i by 5.6 units, Pina-

D1a/Pinb-D1i by 12.6 units, and Pina-D1j/Pinb-D1i by 3.8 units. In examining the

effects of one of these novel loci, Pina-D1c/Pinb-D1h, on end use quality, it is evident

that the synthetic Ha haplotype can decrease sugar snap cookie diameter and increase

flour particle size. Other baking quality traits appear to be unaffected by the Pina-

D1ac/Pinb-D1h Ha haplotype. These findings may be beneficial to breeders looking to

improve the baking qualities of high yielding soft wheat varieties.

CHAPTER 1

INCREASED WHEAT (TRITICUM AESTIVUM L.) GRAIN HARDNESS

CONFERRED BY NOVEL PUROINDOLINE HAPLOTYPES FROM AEGILOPS

TAUSCHII

Introduction

Grain texture is an important factor in the classification of hexaploid (Triticum

aestivum L.) wheat and helps to determine end use quality (reviewed in Morris and Rose,

1996). The Hardness (Ha) locus, the major contributing factor to grain texture, is simply

inherited (Symes, 1965) and is located on the short arm of chromosome 5 (Law et. al.

1978) of all three hexaploid wheat diploid progenitor species (Dubcovsky and Dvorak

1995; Jolly et al. 1996; Sourdille et al. 1996; Giroux and Morris 1997). Bread wheat

evolved from a hybridization of Triticum urartu (AA) and a relative of Aegilops

speltoides (BB), to form Triticum turgidum ssp. dicoccoides (AABB). A subsequent

hybridization of Aegilops tauschii (DD) and a domesticated tetraploid wheat species,

likely Triticum turgidum ssp. dicoccum (AABB; Reviewed in Feldman et. al. 1995),

resulted in common hexaploid wheat (Triticum aestivum L. (AABBDD). Interestingly,

the Ha locus is absent on both the 5A and 5B chromosomes of hexaploid wheat due to a

deletion which occurred during the tetraploidization event and independently and

recurrently among different polyploid wheats (Li et al. 2008). Therefore, introgression of

diverse Ha loci would require new D genome donors. Ha locus diversity exists among A.

tauschii accessions (Massa et. al. 2004). By recreating the hybridization of T. turgidum

2

and A. tauschii (Mujeeb-Kazi et. al. 1996), novel Ha loci can be incorporated into

synthetic wheats and then intercrossed with modern cultivars.

The Ha locus contains 3 linked genes, Puroindoline-a (Pina) (Sourdille et al.

1996; Giroux and Morris, 1998), Puroindoline-b (Pinb) (Giroux and Morris, 1997), and

Grain softness protein (Gsp-1) (Jolly et al. 1996), which are collectively contained within

a 70 kbp DNA segment on the short arm of chromosome 5D (Chantret et al. 2005). The

starch granule bound marker protein for grain softness, friabilin (Greenwell and

Schofield, 1986), is made up of PINA and PINB and there is no direct evidence that GSP-

1 has any effect on grain hardness, despite the tight linkage of the three genes (Giroux

and Morris, 1998). Surveys of Pina and Pinb alleles present in hexaploid wheat varieties

have found that all soft wheats contain the Ha locus haplotype Pina-D1a/Pinb-D1a,

while all hard wheats contain a point mutation in either Pina or Pinb (Giroux and Morris,

1998). Pins account for less than 12% of the hardness differences among hard wheats

(Giroux et al. 2000), but in crosses between hard and soft wheats, they account for nearly

60% of the differences (Campbell et al. 1999). Diverse Pina-D1/Pinb-D1 haplotypes that

give soft grain phenotype would allow for studies of Pin function, and could provide

improved soft wheat milling and baking quality. Novel haplotypes could either be created

via mutagenesis of hexaploid wheats (Feiz et al. 2009a) or from the use of novel D

genome donors. In fact, 19 unique Pina-D1/Pinb-D1 haplotypes were found among 50

A. tauschii and 75 synthetic hexaploid accessions (Massa et al. 2004; Gedye et al. 2004).

Grain hardness was measured on 75 synthetic hexaploids varying for Ha haplotype as a

result of having different A. tauschii donors. Gedye et al. (2004) found two alleles, Pina-

3

D1c and Pinb-D1h, independently were associated with a 9.3 and a 4.6 unit decrease in

hardness, respectively. However, Gedye et al. (2004) did not examine linkage between

the novel Ha haplotypes and grain texture and therefore the observed differences could

have been due to genes unlinked to Ha.

Novel Ha haplotypes could also have an impact on milling and baking quality.

Hard wheats are generally used for breads and soft wheats are generally used for cakes

and cookies (Rogers et al. 1993). The major grain hardness quality associated traits are

those affected by changes in starch damage, particle size, and size distribution after

milling, which lead to different swelling, pasting, and gelling qualities (Dengate, 1984).

Within the hard wheat class, relatively minor (<10 hardness unit) Ha locus mediated

hardness changes impact nearly all milling and baking traits (Martin et al. 2001, 2008).

Within the soft wheat market class, Campbell et al. (2007) found that increasing

puroindoline dosage via chromosome substitution reduced grain hardness and modified

milling properties. Therefore, functional Ha locus haplotypes that alter PINA and/or

PINB functionality could modify both grain hardness and milling and baking quality.

However, novel Ha haplotypes from A. tauschii would likely be of value only in

modifying soft wheat end product quality as the grain hardness values of all tested

synthetic hexaploid wheats are within the soft wheat class (Gedye et. al. 2004)

Here, near isogenic lines were used to further investigate the impact of these

novel haplotypes on grain texture while minimizing other background effects. F2‟s were

then used to test hardness of all four novel haplotypes. The data indicate that all novel Ha

4

haplotypes confer increased grain hardness without significantly affecting the expression

of Pina or Pinb at the transcript or protein level.

Materials and Methods

Plant Material

Nine synthetic wheat accessions were randomly selected from the Kansas State

Wheat Genetic Resource Center (Table 1). Genomic DNA was extracted from an

individual seedling (Riede and Anderson 1996) of each accession with each sequenced

plant being used as the seed source of the novel Ha locus in all subsequent experiments.

Pina and Pinb coding sequences were then amplified from each accession using specific

primers (Table 2) and directly sequenced (SeqWright, Houston, TX) with the

amplification primers following gel purification using a QIAquick Gel Extraction Kit

(Qiagen, Valencia, CA). The PCR cycling parameters consisted of an initial denaturation

of 95ºC for 3 min, followed by 40 cycles of 95ºC for 30 sec, 50ºC for 30 sec, 72ºC for 1

min, and a final extension at 72ºC for 8 min. Crosses were made between elite synthetic

line #14 (Syn14, Table 1) containing the haplotype Pina-D1c/Pinb-D1h and the soft

white spring wheat varieties „Alpowa‟ (PI 566596; 1994 release from Washington State

University) and „Vanna‟ (PI 587199; 1995 release for Western Plant Breeders Inc.); both

of which have the soft wheat Ha locus haplotype Pina-D1a/Pinb-D1a). „Alpowa‟ and

„Vanna‟ were used as the female parent in the first and all subsequent backcrosses. The

„Alpowa‟ x Syn14 population was backcrossed three times and the „Vanna‟ x Syn14

population was backcrossed two times. Selection for the novel Ha haplotype was done in

every generation, starting with selection of heterozygotes in the F1‟s. For the „Alpowa‟ x

5

Syn14 population, Ha locus heterozygotes were followed through the BC3F1 generation

with BC3F2‟s used to determine single gene Ha segregation ratios and to select

homozygous individuals with either the Pina-D1c/Pinb-D1h locus and or the Pina-

D1a/Pinb-D1a locus. For the „Vanna‟ population, Ha locus heterozygotes were followed

through the BC2F1 generation with BC2F2‟s used the same as for the „Alpowa‟

population. Selection was done on DNA from individual BC2F2 and BC3F2 plants,

respectively, while PIN expression levels were measured using BC3F2:3 and BC2F2:3

seeds. Eight additional synthetic wheat accessions (Table 1) with five containing the

Pina-D1c/Pinb-D1h haplotype and one each for the Pina-D1e/Pinb-D1i, the Pina-

D1a/Pinb-D1i and the Pina-D1j/Pinb-D1i haplotypes were crossed with „Vanna‟. F2‟s

were used to determine single gene Ha segregation ratios and to select homozygous

individuals with either the synthetic Ha locus and or the Pina-D1a/Pinb-D1a Ha locus.

Table 1. Synthetic hexploid parental lines and ID‟s based on the Kansas State University Wheat Genetics Resource Center database of

CIMMYT Elite 97 Synthetic Lines

Elite # a Generation

studied

Ha locus haplotype Protein haplotype Cross ID # a

Durum genome

donor a,b

A. tauschii

donor

accession a

7 F2 Pina-D1e/Pinb-D1i PINAc/PINBh CIGM87.2770-1B-

0PR-0B

ALTAR 84 WX205

13 F2 Pina-D1c/Pinb-D1h PINAc/PINBh CIGM88.1209-0B D67.2/P66.270 TA2462

14 BC2F2 and

BC3F2

Pina-D1c/Pinb-D1h PINAc/PINBh CIGM90-561 YUK WX217;TA2

462

26 F2 Pina-D1c/Pinb-D1h PINAc/PINBh CIGM90.525 ACO89 TA2454

57 F2 Pina-D1c/Pinb-D1h PINAc/PINBh CIGM90.812 LCK59.61 TA2460

72 F2 Pina-D1c/Pinb-D1h PINAc/PINBh CIGM90.906 SNIPE/YAV79//DA

CK/TEAL/3

TA2450

74 F2 Pina-D1c/Pinb-D1h PINAc/PINBh CIGM90.910 YAV7 2 / TEZ TA2468

34 F2 Pina-D1j/Pinb-D1i PINAj/PINBh CIGM88.1363-0B DOY1 WX511

29 F2 Pina-D1a/Pinb-D1i PINAa/PINBh CIGM88.1288-0B 68.111/RGB-U

//WARD/3

TA2475

a Information from Kansas State Wheat genetics resource center (http://www.k-state.edu/wgrc/)

b Pedigree of the durum parent with the subsequent cross being to the A. tauschii parent

6

Table 2. Primers used for Pina and Pinb.

No.a

Gene Genbank

accession

Primer Sequences

Forward (5‟-3‟) Reverse (5‟-3‟)

Size of

Product b

1 Pina X69913 ATGAAGGCCCTCTTCCTCA TCACCAGTAATAGCCAATAGTG 446

(25-471)

2 Pinb X69912 ATGAAGACCTTATTCCTCCTA TCACCAGTAATAGCCACTAGGGAA 446

(17-463)

3 Pina X69913 TAGCGAAGTTGTTGGCAGTT TTGAGCATCGATCTAGCAGG 109

(87-196)

4 Pinb X69912 GTTTCACAATGAAGGATTTTCCA TGTGGTGCTATCTGGCTCAG 111

(189-300)

5 Actin c

AF326781 AAATCTGGCATCACACTTTCTAC GTCTCAAACATAATCTGGGTCATC 151

(40-190)

a 1-2, PCR primers (Gautier et al. 1994); 3-4, qRT-PCR primers; 5 expression control primers for qRT-PCR.

b Numbers in the parentheses are positions within the NCBI database accession of primer annealing.

c Gene for Actin expression (ACT-1) used as an expression control in the qRT-PCR experiment

7

8

Selection of Segregating Lines

All synthetic Ha locus haplotypes were distinguished from soft type Ha locus

haplotypes by digesting amplified Pinb coding sequence with BstNI. A master mix of

enzyme/buffer solution consisting of 0.3 µL 10x BstNI enzyme (New England BioLabs,

Ipswich, MA), 0.3 µL enzyme buffer, and 2.4 µL H2O was added to each 20 µL PCR

reaction and the reactions were incubated at 60ºC for 2.5 hrs. Digested PCR products

were electrophoresed on 3% MetaPhore high resolution agarose (Lonza, Rockland, ME)

gels. The Pinb-D1h allele is not digested by BstNI, thus, undigested 447 bp PCR products

were scored as Pinb-D1h alleles. The Pinb-D1a allele is cut once by BstNI resulting in

202 and 245 bp products. Genotypes giving all three possible products after digestion

(202, 245, and 447) were scored as heterozygous for the synthetic Ha locus. A subset of

each genotype were verified by direct sequencing (SeqWright, Houston, TX) of the Pina

and Pinb coding sequences after PCR amplification and gel purification using a

QIAquick Gel Extraction Kit (Qiagen, Valencia, CA).

Field Testing

The two backcross populations were planted as a randomized complete block with

two replications. Each experiment was planted in irrigated and dryland environments in

two years at the Montana State University Arthur H. Post Field Research Center near

Bozeman, MT on 16 May 2008 and 14 May 2009. The eight populations were grown in

randomized block split plot design where main plots were F2 populations and subplots

were individual entries within each cross. The experiment was planted 14 May 2009 at

the same site as the backcross populations. All plots were a single 3m row spaced 30 cm

9

apart seeded with 10 g of seed per plot for the backcross populations and 4 g of seed per

plot for the F2 populations. Prior to harvesting, flowering date was recorded as the days

from planting to anthesis. At maturity, seed from all plots was harvested for further

analysis.

RNA Expression and qRT-PCR

Total RNA was extracted from 4 of the „Vanna‟ background BC2F2 derived lines

(2 Pina-D1c/Pinb-D1h homozygotes and 2 Pina-D1a/Pinb-D1a Ha locus homozygotes)

from immature seeds harvested 21 days postanthesis (dpa) following the procedure

previously described (Hogg et al. 2004). Pure RNA was treated with RQ1 RNase-Free

DNase (Promega, Madison, WI). RT-PCR was performed using a M-MLV reverse

transcriptase kit (Promega, Madison, WI). The cDNA template (100 ng) was added to

the PCR premix specified by the DyNAmo SYBR Green Two-Step qRT-PCR kit

(Finnzymes, Woburn, MA), and expression levels were determined using a MiniOpticon

RT-PCR machine (BioRad, Hercules, CA). For the Pina-D1c/Pinb-D1h and the Pina-

D1a/Pinb-D1a lines, loading controls were prepared using RNA isolated from 21 dpa

seeds of the soft spring wheat „Heron‟ (PI 290910). Specific primers were used for each

gene (Table 2) with Actin used as an expression control gene (Wicker et al. 2001). The

loading controls for each gene were 0.01x, 0.1x, 1x, and 10x, with 1x equaling 100 ng

cDNA template. The fluorescence threshold was set at the level in which the correlation

value of the loading series C(t) value and load values in ng was closest to 1. R2 values

ranged from 0.99 for the Actin loading series curve to 0.93 for the Pina loading series

10

curve. The regression line was used to convert sample C(t) values to a relative cDNA

load value. Pina and Pinb levels were compared to the Actin value for each sample.

Total TX-114 and Friabilin Levels

Total TX-114 extractable puroindoline and starch-surface puroindoline (friabilin)

levels were quantified from mature seed whole meal prepared from „Vanna‟ x Syn14

BC2F2:3 lines that contained either the PINAc/PINBh or the PINAa/PINBa haplotype, 2

F2 populations („Vanna‟ x elite line # 29, with either PINAa/PINBh or PINAa/PINBa, and

„Vanna‟ x elite line #34, with either PINAj/PINBh or PINAa/PINBa), and parental

controls. The PINA null variety, „McNeal‟ (PI 574642; Lanning et al. 1994) and the

PINB null variety, „Canadian Red‟ (CItr 6282; Clark et al. 1926) were also loaded as

experimental controls. For TX-114 extractable puroindoline, seeds were ground with a

mortar and pestle and puroindolines were extracted from 100 mg of whole seed meal as

previously described (Giroux et al. 2003). The extracted proteins were then suspended in

60 µl SDS loading buffer w/o reducing agents (Laemmli, 1970), heated to 70ºC for 10

min and total puroindolines were quantified after electrophoresing on 10 x 10 cm 10-20%

Tris-Glycine acrylamide gels (Lonza, Basel, Switzerland) and staining with Coomassie

Blue Stain (30% methanol, 10% acetic acid, and 1g/L Brilliant Blue R-250). PINA and

PINB were quantified individually after electrophoresing on 14.75% 20 x 20 cm

polyacrylamide (acrylamide:Piperazine Diacrylamide ; 30:0.0135; Lamelli, 1970) gels.

Gels were silver stained (Bloom et. al., 1987), and PIN bands were quantified visually by

comparison with a standard curve created using TX114 proteins extracted from „Vanna‟,

with 1x equaling 8 µL of extractable protein in SDS loading buffer.

11

Friabilin was prepared from starch isolated from 200 mg of mature seed whole

meal as follows. First, 1 mL 0.1 M NaCl was added to each sample in a 1.5 mL tube and

samples were vortexed till suspended before incubating at RT for 30 min. Each sample

was then spun for 10 sec at 13,000 g and the flour was worked into a dough ball with a

pestle. Next, the starch in suspension was pipetted into a new, pre-weighed 2 mL tube

and the dough ball was washed two more times with 0.5 mL 0.1 M NaCl with the

supernatant containing suspended starch transferred each time to the same 2 ml tube. The

starch suspension was then spun at 13,000 g for 1 min and the supernatant was aspirated.

The starch pellet was washed twice by vortexing till suspended in 1 mL 75% w/v

ZnSO4·7H2O, then the samples were centrifuged 1 min at 13,000 g, and the supernatant

was removed by aspiration (Guraya et. al. 2003). The third high salt solution wash was

performed the same as the ZnSO4 washes but used 1 mL 80% w/v CsCl (Guraya et. al.

2003). The starch pellets were then sequentially washed 3 times with 1 mL water and

with 1 mL of acetone by vortexing, centrifuging, and aspirating as before. The starch

pellet was then allowed to dry and the starch weight was recorded. The starch was

resuspended in 400 µL of a 50% isopropanol/0.05 M NaCl solution (Bettge et al. 1995),

vortexed at room temperature for 30 min, and centrifuged for 3 min. at 13,000 g. The

supernatant was then transferred to a new tube and 1.2 mL acetone was added and the

samples were incubated overnight at -20ºC. The samples were then centrifuged for 3 min

at 13,000 g, the supernatants were aspirated and the pellets were washed with acetone and

allowed to dry. For every 100 mg of starch recovered, 240 µL SDS loading buffer w/o

reducing agents (Laemmli, 1970) was added and samples were incubated at 70οC for 10

12

min. PINA and PINB were quantified relative to a loading control series created using a

„Vanna‟ friabilin extract where 1x was equal to 12 µL. Proteins were electrophoresed,

stained, and quantified using the same 14.75% 20 cm x 20 cm gels described above.

Grain Hardness and Seed Size Traits

Grain hardness and seed size traits were measured using the Single Kernel

Characterization System (SKCS) (Perten Instruments, Springfield, IL) with each trait

measured on at least 50 clean, unbroken wheat kernels from each field grown replication

for the backcross and F2 populations.

Data Analysis

For the backcross populations data for each response variable were analyzed via

analysis of variance. The model was that for a randomized complete block design

combined over environments. The entries source of variation was partitioned into

recurrent parent (Alpowa vs Vanna) Puroindoline haplotype (Pina-D1a/Pinb-D1a vs

Pina-D1c/Pinb-D1h), a recurrent parent by Puroindoline haplotype interaction and lines

within recurrent parent-Puroindoline haplotype combination and interactions with

environment. The lines within recurrent parent-Puroindoline haplotype combination were

considered random while other factors were fixed. Analyses were completed using PROC

MIXED in SAS. Each F2 population was analyzed separately using a model for

randomized complete block using PROC MIXED in SAS. The entries source was

partitioned into Puroindoline haplotype and lines within Puroindoline haplotype class.

The lines within Puroindoline class was considered a random effect.

13

Results

Plant and Genetic material

Nine randomly selected accessions of synthetic wheat were obtained from Kansas

State University, and Pina and Pinb were amplified and sequenced using the primers

listed in Table 2. Six were found to carry the Pina-D1c/Pinb-D1h haplotype while one

each possessed the Pina-D1e/Pinb-D1i, Pina-D1a/Pinb-D1i, and the Pina-D1j/Pinb-D1i

haplotypes (Table 1). At the protein level, the Pina-D1c/Pinb-D1h and Pina-D1e/Pinb-

D1i haplotypes both translate to PINAc/PINBh while Pina-D1a/Pinb-D1i translates to

PINAa/PINBh, and Pina-D1j/Pinb-D1i translates to PINAj/PINBh (Figure 1 and Table

1). PINAc contains one amino acid substitution while PINAj shares the same amino acid

substitution and contains an additional substitution relative to PINAa. PINBh contains 14

amino acid substitutions relative to PINBa. Elite Synthetic line #14 carries two novel

alleles, Pina-D1c/Pinb-D1h (PINAc and PINBh), and was chosen to be used in a

backcross program with the soft spring wheat varieties „Alpowa‟ and „Vanna‟ used as

recurrent parents with crossing continued to the BC3F2 and BC2F2 generations,

respectively. The remaining eight synthetic accessions were crossed with „Vanna‟ and the

F2 generation was studied. Ha locus segregation of the BC2F2, BC3F2, and F2 populations

was checked using a Chi-square test and all ratios were consistent with a single gene

1:2:1 segregation ratio (Table 5).

The BC2F2:3 and BC3F2:3 generations were grown in the field in 2008 and

flowering dates were recorded (Appendix). For the BC2F2:3 derived Pina-D1c/Pinb-D1h

lines in the „Vanna‟ background, flowering time did not significantly differ from Pina-

14

D1a/Pinb-D1a. For the BC3F2:3 derived Pina-D1c/Pinb-D1h lines in the „Alpowa‟

background, flowering time did not differ significantly from Pina-D1a/Pinb-D1a lines.

Flowering dates were not recorded for the F2 populations. BC2F2:4 and BC3F2:4

generations were planted in the field in 2009. Agronomic traits were measured including

plant height and yield (Appendix). For the BC2F2:4 derived Pina-D1c/Pinb-D1h lines in

the „Vanna‟ background, plant height and yield did not significantly differ from Pina-

D1a/Pinb-D1a. For the BC3F2:4 derived Pina-D1c/Pinb-D1h lines in the „Alpowa‟

background, plant height and yield did not differ significantly from Pina-D1a/Pinb-D1a

lines.

Puroindoline-a

PINAa – MKALFLIGLLALVASTAFAQYSEVVGSYDVAGGGGAQQCP

PINAc – ****************************************

PINAj – ****************************************

PINAa – VETKLNSCRNYLLDRCSTMKDFPVTWRWWKWWKGGCQELL

PINAc – ****************************************

PINAj – ****************************************

PINAa – GECCSRLGQMPPQCRCNIIQGSIQGDLGGIFGFQRDRASK

PINAc – *****Q**********************************

PINAj – *****Q**********************************

PINAa – VIQEAKNLPPRCNQGPPCNIPGTIGYYW

PINAc – ****************************

PINAj – ***************R************

Fig. 1. Deduced amino acid sequences of both Puroindoline-a and Puroindoline-b from

nucleic acid sequencing of „Vanna‟ as the „soft type‟ PINAa/PINBa parent, elite line #34

as the synthetic PINAj/PINBh parent, and elite lines #7, 13, 26, 57, 72, 74 as the

synthetic PINAc/PINBh parent. An asterisk (*) indicates conserved identity with the wild

type sequence.

15

Figure 1 continued

Puroindoline-b

PINBa– MKTLFLLALLALVASTTFAQYSEVGGWYNEVGGGGGSQQC

PINBh – ********************************A**S****

PINBa – PQERPKLSSCKDYVMERCFTMKDFPVTWPTKWWKGGCEHE

PINBh – *L*************GW********F**************

PINBa – VREKCCKQLSQIAPQCRCDSIRRVIQGRLGGFLGIWRGEV

PINBh – ***N******************GM***K****F*****D*

PINBa – FKQLQRAQSLPSKCNMGADCKFPSGYYW

PINBh – **KI************************

Pina and Pinb Transcript Levels

Pina and Pinb transcript levels were measured using qRT-PCR on RNA isolated

from developing seeds (21 dpa). The mean Pina C(t) values for the BC2F2:3 derived lines

in the „Vanna‟ background were 15.54 for Pina-D1c and 14.75 for Pina-D1a (Table 3).

The relative cDNA amounts for Pina were 0.25 for the Pina-D1c/Pinb-D1h haplotype

and 0.43 for the Pina-D1a/Pinb-D1a haplotype when compared to the „Heron‟ „soft type‟

control. These differences were not significant (Table 3). The mean Pinb C(t) values for

these same lines were 15.69 for Pinb-D1h and 16.80 for Pinb-D1a. The relative cDNA

amounts for Pinb were 0.99 for the Pina-D1c/Pinb-D1h haplotype and 0.43 for the Pina-

D1a/Pinb-D1a haplotype when compared to the „Heron‟ „soft type‟ control. These

differences were also not significant (Table 3).

Table 3. Quantitative real-time PCR C(t) and relative load values of Pina and Pinb for BC2F2 populations.

a Vanna by Pina-D1c/Pinb-D1h. N for both Pina-D1c/Pinb-D1h and Pina-D1a/Pinb-D1a was 2. (Means are given with standard error

in parentheses). Heron was used as a „soft type‟ control. b

C(t) value was determined by the MiniOpticon (BioRad, Hercules, CA) as the point when inflorescence reached a preset threshold. c Relative load value is the relative amount of cDNA compared to a loading curve of 0.01x, 0.1x, 1x, and 10x of „Heron‟ „soft type‟

control. The number represents the Actin 1x value divided by the Actin sample value multiplied by Pin sample value. „Heron‟ values

were given a value of one with the „soft type‟ and synthetic haplotypes compared to them. d

P-value was calculated using a studentized t-test comparing „soft type‟ haplotype means to synthetic haplotype means.

C(t) b

Relative Load c

Haplotype a Pina Pinb Pina Pinb

Vanna (Pina-D1a/Pinb-D1a) x Elite #14 (Pina-D1c/Pinb-D1h)

Heron (Pina-D1a/Pinb-D1a) 14.61 15.59 1 1

Pina-D1c/Pinb-D1h 15.54 (0.31) 15.69 (0.52) 0.25 (0.15) 0.99 (0.33)

Pina-D1a/Pinb-D1a 14.75 (0.30) 16.80 (0.48) 0.43 (0.14) 0.43 (0.10)

P-value d

0.473 0.193 0.499 0.247

17

18

PINA and PINB Protein Expression Levels

To assess the effect of the different Ha haplotypes upon PINA and PINB

expression and degree of starch binding, PINs were extracted from mature seeds with

TX-114 to estimate total extractable PIN and from the surface of starch to assess the

degree of PIN functionality. Total extractable (TX-114) and starch bound levels of

PINA and PINB were then visually assessed using SDS-PAGE (Fig. 2 and 3,

respectively). The null control varieties, „McNeal‟ (PINA null, PINBa) and „Canadian

Red‟ (PINAa, PINB null), were used to show the separation of the PINA and PINB

bands. In the „Vanna‟ parental controls, PINAa is more abundant with its level roughly

twice that of PINBa in terms of both total expression (Fig 2) or as levels bound to starch

(Fig 3). The PINAa/PINBh, PINAc/PINBh, and PINAj/PINBh proteins extracted from

individuals homozygous for the synthetic haplotype show a more abundant band in the

location of the „soft type‟ PINB band with a less abundant band running slightly faster

(Fig 2 and 3). N-terminal sequencing determined that these two bands were in fact PINA

and PINB respectively (results not shown), leading to the conclusion that the novel Ha

PINs have altered mobility but similar expression levels to the PINAa/PINBa proteins of

the „Vanna‟ parental controls or the PINAa/PINBa locus segregants (Fig. 2). The

intensity of these bands was quantified visually and given a value based on the relative

intensity compared to the loading control sequence (Table 4). The „Vanna‟ loading

control bands for total endosperm puroindoline, PINA, and PINB were each set at 1.0 and

the experimental samples were given values in relation to the „Vanna‟ controls.

19

There were no significant differences in either total TX-114 extractable or starch

bound levels of PINA and/or PINB between synthetic allele groups and soft type allele

groups. Levels of PINA in the BC2F2:3 derived lines with the „Vanna‟ recurrent parent

varied up to 20% relative to the wild type controls while in the F2 derived lines with the

PINAa/PINBh haplotype, the mean PINA and PINB values were also within 20% of the

wild type values. In the F2:3 derived lines with the PINAj/PINBh haplotype the mean

TX-114 extractable and starch bound PINA and PINB levels were within 30% of the

control values. While this variation may be enough to cause differences in grain texture,

the differences were not significant. Both PIN bands present for the synthetic Ha loci

appeared lower on the gel than the PIN bands present for the Pina-D1a/Pinb-D1a Ha

locus.

20

Fig. 2. A) TX-114 extracted endosperm puroindolines for „Vanna‟ by PINAc/PINBh

cross. Canadian Red is a PINB null control and McNeal is a PINA null control. There are

three representative independent BC2F2:3 lines shown for the PINAa/PINBa samples and

for the PINAc/PINBh samples in this population. B) TX-114 extracted endosperm

puroindolines for „Vanna‟ by PINAa/PINBh and PINAj/PINBh crosses. There are two

representative independent F2:3 lines for the PINAa/PINBa samples and for the

PINAa/PINBh samples in the Vanna x Syn #29 population, and there are two

representative independent F2:3 lines for the PINAa/PINBa samples and for the

PINAj/PINBh samples in the Vanna x Syn #34 population.

A

21

Fig. 3. A) Friablin extracted from starch showing separated puroindolines for „Vanna‟ by

PINAc/PINBh cross. Canadian Red is a PINB null control and McNeal is a PINA null

control. The same samples were used here as for the total endosperm puroindolines in

figure 2. B) Friabilin extracted from starch showing separated puroindolines for „Vanna‟

by PINAa/PINBh and PINAj/PINBh crosses. The same samples were used here as for the

total endosperm puroindolines in figure 2.

Table 4. Puroindoline expression levels, total puroindoline content, and bound puroindoline content (friabilin) for positive and

negative synthetic lines.

Total PINA b,e

Total PINB b,e

Total PIN b,e

Friabilin PINA c,e

Friabilin

PINB c,e

Total Friabilin c,e

„Vanna‟ a

1 1 1 1 1 1

Haplotype d

BC2F2:3 „Vanna‟ (Pina-D1a/Pinb-D1a) x Elite #14 (Pina-D1c/Pinb-D1h)

PINAc/PINBh 1.2(0.21) 1.3(0.26) 1.3(0.22) 1.0(0.24) 1.0(0.27) 1.0(0.26)

PINAa/PINBa 0.9(0.18) 1.1(0.22) 1.0(0.22) 1.2(0.39) 1.2(0.15) 1.2(0.24)

P-value 0.073 0.383 0.242 0.635 0.243 0.361

F2:3 „Vanna‟ (Pina-D1a/Pinb-D1a) x Elite #29 (Pina-D1a/Pinb-D1i)

PINAa/PINBh 1.2(0.14) 1.1(0.14) 1.2(0.09) 1.0(0.07) 1.0(0.14) 1.0(0.10)

PINAa/PINBa 1.1(0.00) 1.1(0.07) 1.1(0.04) 0.9(0.14) 1.0(0.28) 1.0(0.19)

P-value 0.423 0.698 0.617 0.698 0.998 0.629

F2:3 „Vanna‟ (Pina-D1a/Pinb-D1a) x Elite #34 (Pina-D1j/Pinb-D1i)

PINAj/PINBh 1.3(0.21) 1.2(0.07) 1.2(0.14) 1.00(0.00) 1.0(0.35) 0.97(0.05)

PINAa/PINBa 1.25(0.14) 1.3(0.07) 1.3(0.07) 0.95(0.21) 1.1(0.07) 1.0(0.24)

P-value 0.808 0.293 0.956 0.771 0.733 0.701

a The bands in the „Vanna‟ 1x loading control were given a value of 1 and all other lines were given a value relative to this.

b TX-114 extracted Puroindoline from grain endosperm was quantified using a 0.5x to 2x scale. Number is the average of two

experiments. c Starch bound PINA and PINB was quantified using a 0.5x to 2x scale.

d Results from protein expression of „Vanna‟ by Pina-D1c/Pinb-D1h (BC2F2:3) , Pina-D1a/Pinb-D1i (F2:3) , and Pina-D1j/Pinb-D1h

(F2:3) crosses. e 1.0 was the value assigned to the 1x band for the total, PINA and PINB bands of the „Vanna‟ parent since they are not being

compared to each other in this study. Standard errors are in parentheses. P-value obtained using a studentized t-test.

22

23

Grain Texture

Overall, BC3F2:3 seeds homozygous for the Pina-D1c/Pinb-D1h haplotype in the

„Alpowa‟ background had a mean hardness of 36.6 in 2008 and 33.8 in 2009 and were

significantly harder, by 9.0 and 9.5 SKCS hardness units, respectively, than BC3F2:3 seeds

homozygous for the Pina-D1a/Pinb-D1a haplotype (P=0.020 and P<0.001). Pina-

D1c/Pinb-D1h homozygous seeds in the „Vanna‟ background had a mean hardness of

37.5 in 2008 and 28.1 in 2009 and were also significantly harder (P=0.052 and P<0.001,

repectively) than the Pina-D1a/Pinb-D1a homozygous seeds, which has a mean of 30.2

in 2008 and 16.2 in 2009. In both years, Pina-D1c/Pinb-D1h homozygous seeds showed

no significant difference in kernel weight compared to the „soft type‟ haplotypes in either

background (Table 5).

SKCS hardness was then analyzed on the remaining eight haplotypes in the F2

generation in order to determine if any significant differences in function could be

attributed to the different A. tauschii donors, the different durum donors, or to the

different synthetic haplotypes (Table 6). In all five F2 populations segregating for the

Pina-D1c/Pinb-D1h synthetic haplotype, the synthetic Ha haplotype was associated with

harder texture than seeds with the Pina-D1a/Pinb-D1a haplotype. The differences ranged

from an average of 3.8 units for the „Vanna‟ x Elite line #52 to 7.7 units for the „Vanna‟ x

elite line #26 population (Table 7). While not all five of the differences were significant

at the 0.05 level, when the five populations were combined, the mean hardness for the

Pina-D1c/Pinb-D1h haplotype was 31.0 compared to the mean for the Pina-D1a/Pinb-

D1a haplotype (25.8), was significant (P<0.001). The population segregating for the

24

Pina-D1e/Pinn-D1i haplotype also had a significant difference in SKCS hardness. The

Pina-D1e/Pinn-D1i conferred a 5.6 unit increase over the Pina-D1a/Pinb-D1a haplotype

(P=0.028). The two populations segregating for additional Ha locus haplotypes gave

similar results in that in each case the synthetic Ha locus was associated with increased

grain hardness. In the Vanna x elite synthetic line #34, the Pina-D1j/Pinb-D1h haplotype

group was 3.8 units harder than the seeds with the Pina-D1a/Pinb-D1a haplotype. While

this difference was not significant it did show the same trend as the rest of the

populations. In the Vanna x elite synthetic #29 population, the Pina-D1a/Pinb-D1i group

was 12.6 units harder (P<0.001) than the group having the Pina-D1a/Pinb-D1a

haplotype. No significant differences in kernel weight were seen between the synthetic

and „soft type‟ Ha locus groups in any population (Table 5 and 6). SKCS was also run in

2008 and showed similar results (data not shown).

Table 5. Grain hardness and kernel weight for BC3F2 and BC2F2 derived populations from elite synthetic line #14 (Pina-D1c/Pinb-

D1h) cross with „Alpowa‟ and „Vanna‟ (Pina-D1a/Pinb-D1b) soft white spring wheats.

2008 2009

Ha locus genotype Segregation Data SKCS Grain

Hardness a

Kernel

weight (mg)

a

SKCS Grain

Hardness a

Kernel weight

(mg) a

BC3F2 Alpowa*4/Elite line #14

Pina-D1c / Pinb-D1h 4 36.6

(1.1)

41.0

(0.8)

33.8

(1.0)

35.9

(0.9)

Heterozygote 10 - - - -

Pina-D1a / Pinb-D1a 3 27.6

(1.4)

40.2

(0.7)

24.4

(1.5)

36.4

(0.7)

P-value b,c

0.724 0.020 0.446 <0.001

0.642

BC2F2 Vanna*3/Elite line #14

Pina-D1c / Pinb-D1h 3 37.5

(1.8)

35.0

(0.7)

28.1

(1.2)

36.4

(1.0)

Heterozygote 15 - - - -

Pina-D1a / Pinb-D1a 4 30.2

(2.5)

36.2

(0.9)

16.2

(1.1)

36.4

(0.9)

P-value 0.223 0.052 0.326

<0.001

0.999

a Mean represents average of two replications in two environments with standard error in ().

b P-value for the segregation data is based on a Chi-squared test for single gene segregation model

c P-value for hardness and kernel weight is based on studentized t-test.

25

Table 6. Single Kernel Characterization System (SKCS) and segregation data of F2 populations.

Ha locus genotype Segregation Data a

SKCS Grain

Hardness a

Kernel weight (mg) a

„Vanna‟ x Elite #13 Pina-D1c/Pinb-D1h

Pina-D1c / Pinb-D1h 15 28.2 (1.6) 38.3 (0.9)

Heterozygote 30 - -

Pina-D1a / Pinb-D1a 26 23.9 (1.3) 38.6 (0.9)

P-value b,c

0.078 0.042 0.755


Pina-D1c / Pinb-D1h 11 37.4 (2.1) 35.8 (0.9)

Heterozygote 22 - -

Pina-D1a / Pinb-D1a 14 29.7 (3.1) 35.8 (0.5)

P-value 0.75 0.048 0.142


Pina-D1c / Pinb-D1h 21 25.1 (1.3) 40.7 (0.8)

Heterozygote 41 - -

Pina-D1a / Pinb-D1a 17 21.3 (1.5) 40.1 (0.9)

P-value 0.771 0.063 0.61


Pina-D1c / Pinb-D1h 15 30.8 (2.3) 39.8 (0.7)

Heterozygote 35 - -

Pina-D1a / Pinb-D1a 22 25.4 (2.8) 39.2 (0.9)

P-value 0.492 0.145 0.537


Pina-D1c / Pinb-D1h 13 33.6 (1.5) 39.6 (0.9)

Heterozygote 36 - -

Pina-D1a / Pinb-D1a 14 28.7 (1.4) 39.6 (1.1)

P-value 0.518 0.030 0.989

26

(Table 6 continued)

„Vanna‟ x Pina-D1c/Pinb-D1h Combined

Pina-D1c / Pinb-D1h 75 31.0 (0.8) 38.5 (0.4)

Heterozygote 164 - -

Pina-D1a / Pinb-D1a 93 25.8 (0.9) 38.9 (0.5)

P-value 0.368 <0.001 0.570

„Vanna‟ x Elite #7 Pina-D1e/Pinb-D1i

Pina-D1e / Pinb-D1i 21 32.8 (1.7) 32.6 (0.6)

Heterozygote 37 - -

Pina-D1a / Pinb-D1a 30 27.2 (1.2) 34.1 (0.8)

P-value 0.131 0.028 0.169

„Vanna‟ x Elite #29 Pina-D1a/Pinb-D1i

Pina-D1a / Pinb-D1i 25 39.0 (1.4) 35.2 (0.5)

Heterozygote 39 - -

Pina-D1a / Pinb-D1a 18 26.4 (1.7) 36.3 (0.6)

P-value 0.499 b <0.001

¶ 0.153

¶

„Vanna‟ x Elite #34 Pina-D1j/Pinb-D1i

Pina-D1j / Pinb-D1i 17 28.4 (1.6) 38.6 (0.8)

Heterozygote 35 - -

Pina-D1a / Pinb-D1a 17 24.6 (1.6) 37.8 (1.1)

P-value 0.993 b 0.103 0.568

¶

a Mean represents average of four replications in 2009 with standard error in ().

b P-value for the segregation data is based on a Chi-squared test

c P-value for hardness and kernel weight is based on studentized t-test and analyzed using the PROC MIXED procedure in SAS.

27

Table 7. Hardness differences based on haplotype.

Ha locus haplotype a

SKCS hardness

increase c

Pina-D1c / Pinb-D1h b

5.2a

Pina-D1e / Pinb-D1i 5.6a

Pina-D1j / Pinb-D1i 3.8a

Pina-D1a / Pinb-D1i 12.6b

a Synthetic haplotype that confers increased hardness compared to the „soft type‟ haplotype in the same F2 population.

b Pina-D1c/Pinb-D1h difference represents the mean differences of all five populations with this synthetic haplotype.

c Increase compared to the samples with the Pina-D1a/Pinb-D1a haplotype. Mean differences followed by the same letter do not

differ significantly.

28

29

Discussion

The objective of this study was to test the impact of the Pina-D1c/Pinb-D1h

haplotype in near isogenic lines, and to examine the Pina-D1c/Pinb-D1h haplotype and

other unique Ha haplotuypes in segregating populations. Gedye et al. (2004) reported the

Pina-D1c/Pinb-D1h haplotype was associated with decreased grain hardness. In Gedye‟s

study, the PINA and PINB alleles were described independent of each other and all

hardness testing was done on the original synthetic parents, leading to possible

background effects (Gedye et al. 2004). Here, Pina-D1c/Pinb-D1h was introduced into

common wheat cultivars, which was specifically done by analyzing the haplotypes found

in 9 randomly chosen synthetic hexaploid accessions (Massa et al. 2004), and crossing

them into the soft white spring wheat varieties „Alpowa‟ and „Vanna‟. Sequence analysis

of these accessions revealed 4 novel Ha locus haplotypes (Table 1 and Fig. 1). Seven had

the synthetic protein haplotype PINAc/PINBh, the haplotype previously associated with

increased grain softness in a survey of synthetic wheats (Gedye et al. 2004). By

examining this haplotype in crosses with soft common hexaploid wheat cultivars and in

more molecular detail, the hope was to try to ascertain the true functional effect of this

haplotype. The other two synthetic accessions had the protein haplotypes PINAa/PINBh

and PINAj/PINBh (Table 1 and Figure 1). These haplotypes were also analyzed for their

effects on grain texture. One PINAc/PINBh accession had its Ha locus backcrossed into

two different soft wheat varieties in order to reduce the amount of non-Ha locus linked

genes from the durum or A. tauschii donor. The remaining eight accessions were crossed

30

with the soft white wheat variety, „Vanna‟, and the populations were analyzed as F2‟s.

This was done to try and measure the amount of background effect attributable to the

durum or A. tauschii donors, as well as to test the novel haplotypes, PINAc/PINBh and

PINAj/PINBh.

The effect of the unique Ha locus haplotypes upon Pina and Pinb transcript levels

was assessed with qRT-PCR (Table 3). Previous studies have used northern blot

analysis to quantify RNA expression (Giroux and Morris, 1997). Real-time PCR allows

for higher throughput (Heid et al. 1996). Heid et al. (1996) also showed that equal

amounts of input DNA can give up to a 1 cycle standard deviation, which equates to

almost a 2 fold difference in expression. Therefore, greater than 2-fold increase or

decrease in expression is required for detection. Our results indicated that there was little

to no difference in Pina or Pinb transcript levels between synthetic and „soft type‟ lines

in the BC2F2:3 Pina-D1c/Pinb-D1h population (Table 3).

With no apparent differences in RNA expression, a test for the abundance and

function of PINA and PINB, was a logical subsequent step, and was done on all three of

the Ha haplotypes. By extracting puroindolines from the endosperm of a seed using TX-

114, the absolute level of PINA and PINB in seeds can be assessed (Blochet et al. 1991,

1993; Giroux et al. 2003), while the level of PINA and PINB bound to the surface of

starch gives a measure of their function (Bettge et al. 1995). In this way, the effect of

each unique Ha haplotype upon PIN expression level and function was assessed (Fig. 2

and 3, Table 4). None of the synthetic haplotypes was associated with changes in PIN

expression or starch binding. If small differences were observed in starch binding, it

31

would be predicted that hardness would increase since reduced abundance of either PIN

bound to starch increases grain hardness since both PINs interacting with each other is

necessary to create friabilin (Hogg et al. 2004; Wanjugi et al. 2007; Martin et al. 2007;

Zhang et al. 2009). In this study, it was determined that total friabilin was not

significantly reduced in populations that had the synthetic haplotype (Table 4).

While PIN expression levels were unchanged, each synthetic Ha haplotype

resulted in altered mobility of both PINA and PINB (Fig. 2 and 3). This leads to the

conclusion that the altered mobility results from a conformational change in the synthetic

proteins relative to PINAa and PINBa, which is plausible given that the protein samples

were run on polyacrylamide gels in the absence of reducing agents. Because

puroindolines account for 60% of the hardness differences seen in crosses between hard

and soft wheats (Campbell et al. 1999), it would be plausible that even though no

expression differences were seen, some functional difference may be observed.

This is the first study to report functional differences between different soft Ha

loci. The „soft type‟ wheat cultivars „Alpowa‟ and „Vanna‟ were used as recurrent parents

in crossing with synthetic wheats carrying the Pina-D1c/Pinb-D1h haplotype. Because

the populations were not isogenic, the effects of linked genes upon grain hardness cannot

be excluded. Our results differ from those of Gedye et. al. (2004) who reported that the

Pina-D1c/Pinb-D1h alleles were each associated with softer grain texture, likely due to

other background effects from the durum or A. tauschii donor. Our results differ in

finding that the PINAc/PINBh, PINAj/PINBh, and PINAa/PINBh haplotypes confer a 4

to 13 unit increase in hardness (Table 5 and 6). Severe mutations in PINA or PINB

32

increase SKCS hardness by 30-60 units (Giroux and Morris, 1997, 1998). Soft wheats

typically fall between 24 and 37 hardness units and hard wheats typically fall between 63

and 86 hardness units (Pearson et al. 2009), indicating that the synthetic haplotypes

studied here still confer soft texture. This also indicates some remaining function within

PINA and PINB in all tested Ha locus haplotypes. Interestingly, the difference seen in the

F2 population with the Pina-D1a/Pinb-D1i Ha haplotype shows a much greater increase

in hardness compared to the other populations (Table 7). This leads to the possibility that

the PINBh protein allele is directly responsible for these increases and the PINAc and

PINAj protein alleles may be decreasing hardness. This wasn‟t possible to test because

Pinb-D1a is never paired with a novel Pina allele in any of the available synthetic wheats

(Gedye et al. 2004, Table 1). Presence of friabilin proteins (Fig. 3) helped to support this

idea, as friabilin is scarce in starch isolated from hard wheat varieties (Greenwell and

Schofield, 1986). The relatively small functional differences observed via comparisons

of SKCS grain hardness values indicates that only small differences in expression or

function of the proteins exist, making them difficult to observe. This is consistent with

the fact that we were not able to detect changes in expression at the RNA (Table 3),

protein (Table 4), or starch binding level (Table 4).

Because for the PINAa/PINBh haplotype, the PINA allele is wild type, the

hardness change in that cross or in others can‟t be attributed to PINA but rather to

PINBh. The PINBh allele contains 14 missense mutations relative to the PINBa allele.

Slight decreases in PINB function are more likely to be observed since PINB limits grain

softness more than PINA (Swan et. al. 2006). Overexpression of both PINA and PINB

33

has shown increases in lipid content (Feiz et al. 2009b). PINBh may limit grain softness

via decreases in lipid binding since puroindolines interaction with starch is mediated by

lipids (Greenblatt et al. 1995). Additional experimentation is required to investigate the

effect of different synthetic wheat derived Ha haplotypes upon starch lipid levels.

Conclusion

Because each of the synthetic wheat derived Ha locus haplotypes increased grain

hardness, they could prove useful in modifying soft wheat milling and baking quality.

Testing the BC2F2 population for milling and baking qualities, such as flour yield, starch

damage, ash content, solvent retention and cookie diameter would give some insight into

end use possibilities of these synthetic haplotypes. The creation of near-isogenic lines in

several soft wheat backgrounds could also give more data as to the true effect of these

novel haplotypes. This study has found several synthetic wheat derived Ha locus

haplotypes which increase grain hardness of soft wheat. In particular, the Pinb-D1h

allele is associated with increased grain hardness. Further experiments into the effects of

these novel synthetic Ha loci on milling and baking quality could provide valuable

information for practical use.

34

CHAPTER 2

NOVEL HA LOCUS, PINA-D1C/PINB-D1H, IMPACTS SOFT WHITE SPRING

WHEAT MILLING AND BAKING CHARACTERISTICS

Introduction

Classification of hexaploid wheat (Triticum aestivum L.) varieties depends a great

deal on grain hardness (reviewed in Morris and Rose, 1996). The Ha locus, which

contains three linked genes, Puroindoline-a (Pina) (Sourdille et al. 1996; Giroux and

Morris, 1998), Puroindoline-b (Pinb) (Giroux and Morris, 1997), and Grain softness

protein (Gsp-1) (Jolly et al. 1996), is responsible for 60% of hardness differences

between soft and hard wheats (Campbell et al. 1999). Surveys of Pina and Pinb alleles

present in hexaploid wheat varieties have found that all soft wheats contain the Ha locus

haplotype Pina-D1a/Pinb-D1a, while all hard wheats contain a point mutation in either

Pina or Pinb (Giroux and Morris, 1998). Hard wheats that vary in Pin alleles vary in

grain hardness and end product quality (Martin et al. 2001, 2008). Therefore, in order to

improve soft wheat quality it is important to have a great deal of Ha locus diversity. A

survey of the diploid wheat relative, Aegilops tauschii, found nine new Pina-D1/Pinb-D1

haplotypes (Massa et al. 2004). These haplotypes can also be found in synthetic

accessions of hexaploid wheat, created via the hybridization between the tetraploid

Triticum turgidum (AABB) and A. tauschii (DD; Mujeeb-Kazi et al. 1996). A synthetic

wheat survey of four of these new haplotypes (Pina-D1c/Pinb-D1h, Pina-D1e/Pinb-D1i,

Pina-D1a/Pinb-D1i, and Pina-D1j/Pinb-D1i) demonstrated that all four are functional Ha

loci that confer soft phenotypes (Gedye et al. 2004). In crosses with hexaploid wheats

35

each of the new Ha haplotypes increased grain hardness with differences ranging from 4-

12 Single Kernel Characterization System (SKCS) units versus the Pina-D1a/Pinb-D1a

haplotype (Chapter 1).

Soft wheat is generally used in making cakes and cookies while hard wheat is

generally used for making bread (Rogers et al. 1993). Bread quality is often related to the

amount and quality of protein that is present, and specifically to the high molecular

weight glutenins (Payne et al. 1984). It has also been shown that phospholipids and

glycolipids can increase bread loaf volume (MacRitchie and Gras, 1973), and it is

believed that the PIN proteins are involved in the binding of these lipids to the surface of

starch granules (Greenblatt et al. 1995; Feiz et al. 2009b). Besides lipid binding, the

major grain hardness quality associated traits for soft wheats include starch damage,

particle size and size distribution after milling, which affect swelling, pasting, and gelling

qualities (Dengate et al. 1984). Ultimately, increasing softness among soft wheats is

associated with increased cookie diameter and decreased sucrose retention capacity

(Gaines, 2000; Guttieri et al. 2001). The novel Ha loci from synthetic wheat accessions

are some of the only naturally occurring functional Ha loci and represent an opportunity

to better understand the effects of minor hardness variation within soft wheats upon soft

wheat milling and baking quality.

Here, synthetic wheat haplotype Pina-D1c/Pinb-D1h was backcrossed into two

soft white spring wheat varieties. The two populations were then analyzed for their

milling qualities by measuring their protein content and flour particle size distribution

after milling. End product quality of flour was then analyzed by solvent retention

36

capacities (SRC, AACC method 56-11; Gaines, 2000; Slade and Levine, 1994), and

alkaline water retention capacity (AWRC, AACC method 56-10). Then baking quality

was tested by analyzing sugar snap cookie diameters, heights, and appearance.

Materials and Methods

Plant Material

Seeds of elite synthetic line #14, which has the Ha haplotype Pina-D1c/Pinb-

D1h, were obtained from the Kansas State Wheat Genetic Resource Center and planted in

the greenhouse along with the soft white spring wheat varieties „Alpowa‟ (PI 566596)

(Released in 1994 by Washington State University) and „Vanna‟ (PI 587199) (Released

in 1995 by Western Plant Breeders Inc), both of which have the Ha haplotype Pina-

D1a/Pinb-D1a. Initial crosses were made with „Alpowa‟ and „Vanna‟ as the female

parents and the synthetic line as the male parent.

Selection of Segregating Lines

F1‟s were planted and DNA was extracted from leaf tissue of individual plants

(Riede and Anderson, 1996). DNA was screened by amplifying only Pinb (Primers in

Table 1) digesting the product with BstNI, and running the fragments on high resolution

agarose gels (Reynolds et al. 2009) in order to identify Pinb-D1a/Pinb-D1h heterozygotes

for crossing. Subsequently, the „Alpowa‟ population was backcrossed 3 more times, and

the „Vanna‟ population was backcrossed 2 more times, each with the soft white spring

wheat as the recurrent parent. After each cross, F1‟s were screened to identify Ha locus

heterozygotes. Both populations were then allowed to self and BC3F2‟s and BC2F2‟s were

37

screened for individuals homozygous for either the Pina-D1a/Pinb-D1a or the Pina-

D1c/Pinb-D1h locus (Reynolds et al. 2009). All BC3F2‟s and BC2F2‟s Ha loci were also

verified by direct sequencing (SeqWright, Houston, TX) of the Pina and Pinb coding

sequences (Primers in Table 1) after PCR amplification and gel purification using a

QIAquick Gel Extraction Kit (Qiagen, Valencia, CA).

Field Testing

The two backcross populations were evaluated in separate adjacent field trials.

Each experiment was a randomized complete block with two replications. All plots were

a single 3m row spaced 30 cm apart seeded with 10 g of seed. Each experiment was

planted in irrigated and dryland environments at the Montana State University Arthur H.

Post Field Research Center near Bozeman, MT on 14 May 2009. The irrigated

environment received an additional 6.25 cm of water two weeks prior to anthesis. At

maturity, seed from all plots was harvested for milling and baking analyses.

End Quality Testing

Three lines with the Pina-D1a/Pinb-D1a and four with the Pina-D1c/Pinb-D1h

Ha locus haplotypes from the „Alpowa‟ population from all four replications were tested

for hardness using the Single Kernel Characterization System (SKCS, Perten Instruments,

Springfield, IL). The same was done for 4 lines with Pina-D1a/Pinb-D1a and 3 lines with

Pina-D1c/Pinb-D1h Ha locus haplotypes from the „Vanna‟ population from all four

replications. One hundred grams of seed were then tempered to 14% moisture 18 to 24

hours before being milled on a Quadramat Jr. mill (C.W. Brabender Instrument, Inc.,

South Hackensack, NJ). Bran and flour were weighed individually and flour yield was

38

calculated as the proportion of break flour and reduction flour to total products. Then,

flour was separated using 150, 75, and 53 µm USA standard sifting screens and the sieve

products were weighed. Near-infrared reflectance (NIR, InfraTec GmbH, Dresden,

Germany) was run on the flour to test protein (which is converted from nitrogen content

by multiplying by 6.25), moisture, and ash content.

Flour solvent retention capacities (SRCs, AACC Method 56-11, Slade and

Levine, 1994; alkaline water retention capacity, AACC Method 56-10) were also

measured. The five solvents were deionized water, 50% (w/w) sucrose solution, 5%

(w/w) sodium carbonate solution, 5% (w/w) lactic acid solution, and an alkaline water

solution (0.1 N sodium bicarbonate). The following modifications were made to the

approved AACC method. After pre-weighing 2 mL microcentrifuge tubes, 200 mg of

flour was weighed out and added to each tube along with 1 mL of solvent. The tubes

were vortexed for 2 min in order to disperse the flour, and then the samples were rested

for 18 minutes with 5 seconds of vigorous shaking at 5, 10, 15, and 20 minutes after the

addition of the solvent. After 20 minutes, the tubes were centrifuged for 15 min at 1000 x

g. The supernatants were decanted and the tubes were drained at a 90ο angle for 10 min.

SRC values were calculated as described in AACC method 56-11.

Baking quality was tested using the AACC sugar snap cookie baking procedure

(AACC Method 10-52). Quality was determined through the measurement of four

characteristics; cookie spread (average diameter of two cookies); cookie height (stack

height of two cookies); weight loss upon baking, and top grain score, or the extent of

islanding on the cookie surface compared to a standard set of cookies ranging from a

39

score of 0 to 9. Cookies given a score of 0 have smooth surface indicating poor baking

quality. Islanding, and with it baking quality, increases as the top grain score increases.

Data Analysis

Data for each response variable were analyzed via analysis of variance. The

model was that for a randomized complete block design combined over environments.

The entries source of variation was partitioned into recurrent parent („Alpowa‟ vs

„Vanna‟), Puroindoline haplotype (Pina-D1a/Pinb-D1a vs Pina-D1c/Pinb-D1h), a

recurrent parent by Puroindoline allelic class interaction, lines within recurrent parent-

Puroindoline haplotype combination, and interactions with environment. The lines within

recurrent parent-Puroindoline allelic class combination were considered random while

other factors were fixed. Analyses were completed using PROC MIXED in SAS.

Results

The soft white spring wheat varieties „Alpowa‟ and „Vanna‟ having the Pina-

D1a/Pinb-D1a Ha haplotype were used as recurrent parents in crosses with Elite

Synthetic #14 having the Pina-D1c/Pinb-D1h haplotype. Individual BC3F2 („Alpowa‟

parent) and BC2F2 („Vanna‟ parent) lines were genotyped by amplifying Pinb and

digesting with BstNI to identify individual plants homozygous for either of the two

segregating Ha haplotypes. The Ha haplotypes of all lines were confirmed by sequencing

of Pina and Pinb coding sequences. Ha locus segregation in both the „Alpowa‟ (total of

17 individuals tested) and „Vanna‟ (total of 22 individuals tested) backgrounds was not

significantly different from the expected 1:2:1 ratio for normal single gene segregation as

40

determined by a Chi-square test. In the „Alpowa‟ background population, there were 4

Pina-D1a/Pinb-D1a and 3 Pina-D1c/Pinb-D1h Ha locus homozygotes. In the „Vanna‟

population there were 3 Pina-D1c/Pinb-D1h and 4 Pina-D1a/Pinb-D1a Ha locus

homozygotes. All lines were grown in the field in replicated plots in 2009.

Grain hardness and kernel weight were measured using the Single Kernel

Characterization System (SKCS) and protein content by NIR. While Ha haplotype was

not associated with changes in kernel weight or protein content, it was associated with

changes in grain hardness in both recurrent parent populations (Table 8). In the „Alpowa‟

population, there was a difference of 9.4 units (P<0.001), and in the „Vanna‟ population

there was a difference of 12.0 units (P<0.001) with the Pina-D1c/Pinb-D1h haplotype

associated with increased grain hardness in both populations. Overall, seeds with the

Pina-D1c/Pinb-D1h haplotype were 11.7 units harder than seeds with the Pina-

D1a/Pinb-D1a haplotype (P<0.001).

41

Table 8. Grain hardness, seed weight and protein content for populations of elite

synthetic line #14 (Pina-D1c/Pinb-D1h) cross with recurrent parents „Alpowa‟ (BC3F2;

Pina-D1a/Pinb-D1a) and „Vanna‟ (BC2F2 ; Pina-D1a/Pinb-D1a) soft white spring

wheats.

Haplotypeb # of lines

SKCS

hardnessc

Kernel

Weight (mg)c

Seed Protein

(g/kg)d

„Alpowa‟*4/Elite #14

Pina-D1a/Pinb-D1a 3 24.4 36.4 116.9

se 1.5 0.7 1.1

Pina-D1c/Pinb-D1h 4 33.8 35.9 114.6

se 1.0 0.9 1

P-valuea <0.001 0.642 0.147

„Vanna‟*3/Elite #14

Pina-D1a/Pinb-D1a 4 16.2 36.4 124.3

se 1.1 0.9 1

Pina-D1c/Pinb-D1h 3 28.1 36.4 125.8

se 1.2 1.0 2.2

P-value <0.001 0.999 0.476

Mean

Pina-D1a/Pinb-D1a 7 19.7 36.4 121.1

se 1.2 0.6 1

Pina-D1c/Pinb-D1h 7 31.3 36.1 119.4

se 0.9 0.6 1.5

P-value <0.001 0.72 0.36

Parents

„Alpowa‟ 1 28.1 33.4 116.5

se 0.8 2.1 0.1

„Vanna‟ 1 13.4 28.6 111.3

se 2.3 0.7 0.1

Elite #14 1 37.8 46.8 149.8

se

3.3 2.8 0.2

a P-values determined using a studentized t-test for SKCS hardness, Kernel weight, seed

and flour protein. b

Pina-D1a/Pinb-D1a is the „soft type‟ Ha haplotype and Pina-D1c/Pinb-D1h is the

synthetic Ha haplotype. c Kernel hardness and kernel weight were measured using the single kernel

characterization system. d Whole grain protein was measured using near infrared reflectance (determined by

multiplying nitrogen content by 6.25).

42

For flour yield, only the „Alpowa‟ population showed a significant difference,

with the Pina-D1c/Pinb-D1h lines yielding 1.5% more flour than the „soft type‟ lines

(Table 9). Ash content also trended toward having more in the Pina-D1c/Pinb-D1h lines

than in the Pina-D1a/Pinb-D1a lines, with the differences in the „Vanna‟ population and

the combined means of the two populations being significant (P=0.040 and P=0.024,

respectively). Bran and flour amounts didn‟t differ significantly between the two

haplotypes for either population. For particle sizes, there are differences that appear to be

caused by the Pina-D1c/Pinb-D1h haplotype (Table 10). The Pina-D1a/Pinb-D1a

haplotype had more very small (<53 µm) particles (33.5% vs 30.8% in the „Alpowa‟

population, 33.8% vs 28.6% in the „Vanna‟ populations, and 33.7% vs 29.7% overall,

P=<0.001 in all three cases) and fewer large (>75µm) particles (55.7% vs 58.6% in the

„Alpowa‟ population, 54.9% vs 60.9% in the „Vanna‟ population, and 55.3% vs 59.6%

overall (P= <0.001). The lone exception was for particles greater than 150 µm in the

„Alpowa‟ population. These differences in flour particle size distribution point out

differences in milling properties conferred by these different haplotypes. There was no

significant difference in the amount of flour in the size range between 53 and 75 µm for

either of the populations. There were no noticeable trends for the affects of the novel Ha

locus on SRC and AWRC (Table 11).

43

Table 9. Mean bran and flour yields after milling and flour ash content of synthetic versus

„soft type‟ Ha locus haplotypes. [Elite synthetic line #14 (Pina-D1c/Pinb-D1h) cross with

recurrent parents „Alpowa‟ (BC3F2; Pina-D1a/Pinb-D1a) and „Vanna‟ (BC2F2 ; Pina-

D1a/Pinb-D1a).]

Haplotype b % Bran

c

% Flour

yield c

Flour

Protein

(g/kg) d Ash (g)

e


Pina-D1a/Pinb-D1a 31.3 68.8 114 0.4

se 1.1 0.6 4.1 0.0

Pina-D1c/Pinb-D1h 29.7 70.3 118 0.4

se 0.6 0.3 5.2 0.0

P-value a 0.144 0.021 0.517 0.201


Pina-D1a/Pinb-D1a 29.2 70.8 119 0.4

se 0.5 0.3 5.0 0.0

Pina-D1c/Pinb-D1h 28.8 71.3 121 0.5

se 0.7 0.4 6.4 0.0

P-value 0.437 0.476 0.767 0.040

Mean

Pina-D1a/Pinb-D1a 30.1 70.0 117 0.4

se 0.6 0.4 2.9 0.0

Pina-D1c/Pinb-D1h 29.3 70.7 120 0.5

se 0.5 0.3 3.7 0.0

P-value 0.253 0.037 0.531 0.024

Parents

„Alpowa‟ 30.3 69.8 104 0.4

se 2.5 1.8 1.1 0.0

„Vanna‟ 30.3 69.7 98 0.4

se 0.7 0.3 0.8 0.0

Elite #14 29.8 70.3 145 0.4

se 1.5 0.5 3.1 0.0

a P-values determined using a studentized t-test.

b Pina-D1a/Pinb-D1a is the „soft type‟ Ha haplotype and Pina-D1c/Pinb-D1h is the

synthetic Ha haplotype. c Bran and flour were weighed after milling but prior to sifting.

d Flour protein measured with NIR (determined by multiplying nitrogen content by 6.25).

e Flour ash was estimated using near infrared reflectance and calculated as number of

grams of ash in 100 g of flour.

44

Table 10. Flour particle size distribution of synthetic versus „soft type‟ Ha locus

haplotypes. [Elite synthetic line #14 (Pina-D1c/Pinb-D1h) cross with recurrent parents

„Alpowa‟ (BC3F2; Pina-D1a/Pinb-D1a) and „Vanna‟ (BC2F2 ; Pina-D1a/Pinb-D1a).]

Haplotype b

% >150

µm c

% <150 -

>75 d

%<75 -

>53 e % <53

f


Pina-D1a/Pinb-D1a 26.3 29.4 10.8 33.5

se 0.3 0.3 0.3 0.4

Pina-D1c/Pinb-D1h 26.5 32.1 10.6 30.8

se 0.3 0.2 0.2 0.5

P-value a 0.596 <0.001 0.429 <0.001


Pina-D1a/Pinb-D1a 24.2 30.7 10.5 33.8

se 0.3 0.4 0.7 0.5

Pina-D1c/Pinb-D1h 26.3 34.6 10.5 28.6

se 0.4 0.4 0.3 0.5

P-value <0.001 <0.001 0.941 <0.001

Mean

Pina-D1a/Pinb-D1a 25.1 30.2 10.7 33.7

se 0.3 0.3 0.2 0.4

Pina-D1c/Pinb-D1h 26.4 33.2 10.5 29.9

se 0.2 0.3 0.2 0.4

P-value 0.006 <0.001 0.631 <0.001

Parents

„Alpowa‟ 27.0 30.1 11.6 32.0

se 0.8 0.5 0.5 1.1

„Vanna‟ 21.1 26.8 14.3 37.9

se 0.2 0.6 0.7 0.6

Elite #14 32.5 38.3 9.9 19.3

se 0.6 0.9 0.2 1.01



synthetic Ha haplotype. c Percent of flour particles larger than 150 µm.

d Percent of flour particles between 150 µm and 75 µm.

e Percent of flour particles between 75 µm and 53 µm.

f Percent of flour particles smaller than 53 µm.

45

Table 11. Mean solvent retention capacity percentages for the Pina-D1a/Pinb-D1a „soft

type‟ Ha locus haplotype compared to the Pina-D1c/Pinb-D1h synthetic Ha locus

haplotype. [Elite synthetic line #14 (Pina-D1c/Pinb-D1h) cross with recurrent parents

„Alpowa‟ (BC3F2; Pina-D1a/Pinb-D1a) and „Vanna‟ (BC2F2 ; Pina-D1a/Pinb-D1a).]

Solvent Retention Capacity

Haplotype b

% Sucrose c

% Sodium

Carbonate c

% Alkaline

Water d

% Lactic

Acid c % Water

c

Alpowa*4/Elite #14

Pina-D1a/Pinb-D1a 101.2 89.8 76.6 152.6 72.8

se 0.8 2.1 1.2 2.2 0.8

Pina-D1c/Pinb-D1h 101.0 87.2 75.2 149.2 73.5

se 0.6 1.6 0.6 2.3 0.4

P-value a 0.867 0.317 0.256 0.305 0.374

Vanna*3/Elite #14

Pina-D1a/Pinb-D1a 97.8 82.1 72.8 119.8 68.2

se 0.9 1.2 0.8 3.0 1.1

Pina-D1c/Pinb-D1h 97.4 79.5 71.0 122.3 66.5

se 0.8 1.4 0.7 4.4 0.7

P-value 0.722 0.157 0.115 0.624 0.222

Mean

Pina-D1a/Pinb-D1a 99.5 85.4 74.4 133.8 70.2

se 0.8 1.3 0.8 3.7 0.8

Pina-D1c/Pinb-D1h 99.2 83.9 73.4 137.7 70.5

se 0.7 1.3 0.6 3.4 0.7

P-value 0.773 0.414 0.289 0.448 0.758

Parents

Alpowa 93.8 90.0 80.3 150.6 73.4

se 10.1 2.8 0.6 1.9 0.9

Vanna 103.4 85.7 76.5 125.7 71.8

se 1.9 3.4 0.6 4.0 0.6

Elite #14 108.9 76.8 70.3 103.4 66.6

se 3.7 1.1 1.17 2.8 0.9



synthetic Ha haplotype. c Determined using AACC method 56-11. Calculated as the amount of solvent retained by

the flour as a percentage of the original flour weight at a 14% flour moisture level. d Determined using AACC method 56-10. Calculated as the amount of solvent retained

by the flour as a percentage of the original flour weight at a 14% flour moisture level.

46

Cookie quality was also impacted by Ha locus associated differences (Table 12).

Cookie diameter was smaller with the Pina-D1c/Pinb-D1h synthetic Ha locus haplotype

in the „Vanna‟ populations and overall (P<0.001 for the „Vanna‟ population; and

P=0.020 for the two combined). Cookie height was greater for the Pina-D1c/Pinb-D1h

haplotype for the Vanna population and when combined over the two populations

(P=0.040 for the „Vanna‟ population; and P=0.034 for the two combined). Cookie weight

loss and top grain score showed no significant differences between the differing Ha loci

(Table 12 and Fig.4).

Table 12. Baking quality data represented as means comparing the Pina-D1a/Pinb-D1a

„soft type‟ haplotype to the Pina-D1c/Pinb-D1h synthetic haplotype. [Elite synthetic line

#14 (Pina-D1c/Pinb-D1h) cross with recurrent parents „Alpowa‟ (BC3F2; Pina-

D1a/Pinb-D1a) and „Vanna‟ (BC2F2 ; Pina-D1a/Pinb-D1a).]

Haplotype b

Diameter

(cm) c

Weight

loss (g) d

Cookie

height (cm) c

Top grain

score (0-9) e

Alpowa*3/Elite #14

Pina-D1a/Pinb-D1a 7.98 4.6 0.90 2.4

se 0.0 0.0 0.0 0.3

Pina-D1c/Pinb-D1h 7.94 4.4 0.93 3.2

se 0.0 0.1 0.0 0.3

P-value a 0.592 0.064 0.293 0.269

Vanna*2/Elite #14

Pina-D1a/Pinb-D1a 8.25 4.9 0.87 3.0

se 0.0 0.1 0.0 0.4

Pina-D1c/Pinb-D1h 8.04 4.7 0.93 2.1

se 0.0 0.1 0.0 0.4

P-value <0.001 0.239 0.040 0.808

Mean

Pina-D1a/Pinb-D1a 8.11 4.8 0.89 2.9

se 0.0 0.1 0.0 0.2

Pina-D1c/Pinb-D1h 8.00 4.5 0.93 3.1

se 0.0 0.1 0.0 0.3

P-value 0.020 0.040 0.034 0.522

47

(Table 12 continued)

Parents

Alpowa' 8.02 5.2 0.89 2.8

se 0.0 0.2 0.0 0.4

Vanna' 8.18 4.7 0.92 3.8

se 0.0 0.1 0.0 0.6

Elite #14 8.04 4.5 0.91 2.1

se 0.1 0.2 0.0 0.3



synthetic Ha haplotype. c Cookie diameter and cookie height is measured using two cookies made with the same

flour side by side or one on top of the other and dividing by 2. d Weight loss was calculated by weighing the cookies on the pan before and after they

were baked and finding the difference. e Top grain score is determined through the use of a standardized set of cookies with 9

being the best.

48

Figure 4. Picture of the top of the two flour controls „Blue Mountain‟, Elite line 14, along

with three cookies from the „Alpowa‟ population and three cookies form the „Vanna‟

population. Each population is represented by a „soft type‟ parent, one sample with the

synthetic Ha locus haplotype and one sample with the „soft type‟ Ha locus haplotype.

„Blue Mountain‟ is used as a control with known cookie baking quality and is given a top

grain score of 6. [Elite synthetic line #14 (Pina-D1c/Pinb-D1h) cross with recurrent

parents „Alpowa‟ (BC3F2; Pina-D1a/Pinb-D1a) and „Vanna‟ (BC2F2 ; Pina-D1a/Pinb-

D1a).]

49

Discussion

The objective of this study was to determine the milling and baking quality effects

associated with the Pina-D1c/Pinb-D1h Ha locus haplotype, which confers a harder grain

phenotype than the Pina-D1a/Pinb-D1a haplotype (Reynolds et al. 2009). This was done

by introgressing the Ha haplotype Pina-D1c/Pinb-D1h into the soft white spring wheat

cultivars „Alpowa‟ and „Vanna‟. Previously, these populations have been tested for grain

hardness, showing an average of an 10.6 SKCS unit increase (Table 8), yet showed no

differences in the expression of PIN proteins or RNA transcripts (Chapter 1). Logically,

the next step was to test whether the Pina-D1c/Pinb-D1h Ha haplotype is associated

with changes in soft wheat milling and baking quality.

The first characteristics that were analyzed were milling and flour properties.

Flour protein was measured in order to better predict how these flours would bake into

cookies, and no significant difference was found for this characteristic. Flour ash does

show an increase in the Pina-D1c/Pinb-D1h haplotype (Table 9). Flour ash is used as an

indicator of flour grade color (Symons and Dexter, 1991), but more importantly for this

study it correlates with grain hardness, as shown in lines with extremely low ash content

as a result of transgenic overexpression of puroindolines (Hogg et al. 2005). Here, overall

flour yield is affected only in the „Alpowa‟ population (Table 9), but it is clear that there

is a greater proportion of smaller particles for the Pina-D1a/Pinb-D1a than the Pina-

D1c/Pinb-D1h haplotype in both the „Alpowa‟ and the „Vanna‟ populations (Table 10).

This agrees with previous findings that softer grain texture results in smaller particle size

(Gaines, 1985). Smaller particle size often result in more water absorption, either caused

50

by increased starch damage during particle size reduction (Yamazaki, 1959), or by

increased surface area (Gaines, 1985). The sodium carbonate retention capacity and

alkaline water retention tests can give insight into the amount of flour starch damage and

can be used to predict end product quality differences (Gaines, 2000; Slade and Levine,

1994). Here, both tests indicate that the two tested Ha loci do not affect solvent retention

capacities (Table 11), which would mean that starch damage is not a factor in the smaller

particle size, and will not be a factor to consider in any other end product quality

differences. This could be explained by the fact that soft wheats suffer from less starch

damage than hard wheats (Slaughter et al. 1992) and, therefore, the slight differences in

hardness seen between the Pina-D1a/Pinb-D1a and the Pina-D1c/Pinb-D1h represent a

smaller range than between hard and soft wheat classes. Sucrose retention is indicative of

pentosan and gliadin impact on the absorption of water and, along with protein content, is

highly correlated with sugar snap cookie diameter (Gaines, 2000; Guttieri et al. 2001;

Guttieri and Souza, 2003; Roccia et al. 2006). Sucrose retention capacity has also been

shown to be correlated with sodium carbonate retention capacity (Guttieri et al. 2001).

Here, sucrose retention was unaffected by Ha haplotype (Table 11), which would indicate

that pentosans and/or gliadins are not responsible for increased water absorption. Lactic

acid retention points to glutenins as the reason for increased water absorption (Gaines,

2000). Here, lactic acid retention is also unaffected by the Pina-D1c/Pinb-D1h haplotype,

but there is clearly a difference between the „Alpowa‟ and „Vanna‟ populations. Finding

an increase like this is not surprising because „Alpowa‟ is known to have the 5+10

subunits of the high molecular weight glutenin genes, while „Vanna‟ does not (Park et al.

51

2003). Therefore, the differences seen in lactic acid retention are not a product of the Ha

locus, which is what is being tested here.

Water absorption has an underlying effect on the baking quality of wheat flour.

As explained above, the water absorption in these samples could be caused by differences

in particle size or starch damage, or by the presence of pentosans, gliadins, or glutenins.

Upon baking sugar snap cookies from the flour, cookie diameter was higher in the Pina-

D1a/Pinb-D1a haplotype (Table 12). In contrast, cookie height was smaller in the Pina-

D1a/Pinb-D1a Ha haplotype. High levels of water absorption reduce the amount of

spreading when cookies are baked (Gaines, 2000). The differences seen here could be

explained by the low level of variability, fractions of a millimeter, in the measurements.

It appears that all genotypes have a high degree of water absorption which would lead to

their relatively poor top grain scores (Figure 4). An interesting aspect of the cookie

baking was the differences between the „Alpowa‟ and „Vanna‟ populations. Again, these

are most likely caused by the 5+10 subunits of the high molecular weight glutenins in

„Alpowa‟, which would increase water absorption and decrease cookie spread and top

grain score.

Conclusion

Here, we have demonstrated that the novel Ha locus Pina-D1c/Pinb-D1h confers

larger flour particle size, which leads to smaller sugar snap cookies. The data shown here

would suggest that breeders would not be interested in this novel haplotype because there

is no improvement in baking quality. It is possible, however, that this minor change in

52

end product quality be beneficial in breeding programs. If these lines were grown in a

more appropriate environment, it would be possible that there is a more significant

difference in cookie spread and top grain score than we see here. These findings are also

helpful in the further understanding of the function of the Pin genes in the plant overall.

53

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59

APPENDIX A

BACKCROSS POPULATION AGRONOMIC DATA

60

Haplotype Plant Height

(cm) a

Yield

(kg/row) a,b

Flowering

Time (days) c

Alpowa*4/Elite #14

Pina-D1a/Pinb-D1a 83 581 67.83

se 0.9 1.8 0.30

Pina-D1c/Pinb-D1h 84 562 68.14

se 0.3 3.4 0.21

P-value 0.531 0.641 0.391

Vanna*3/Elite #14

Pina-D1a/Pinb-D1a 83 549 68.79

se 1.0 6.3 0.53

Pina-D1c/Pinb-D1h 84 625 69.09

se 2.1 2.2 0.56

P-value 0.334 0.316 0.724 a Plant height, row length, and yield were measured in 2009 only due to hail in 2008.

b Yield is the average grain from two row plots.

c Flowering time was measured in 2008 and is reported as the number of days between

planting and anthesis.

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INCREASED WHEAT (TRITICUM AESTIVUM L.) GRAIN HARDNESS …